Tumor necrosis factor a (TNF-.alpha. also cachectin) is an important cytokine that plays a role in host defense. The cytokine is produced primarily in macrophages and monocytes in response to infection, invasion, injury, or inflammation. Some examples of inducers of TNF-.alpha. include bacterial endotoxins, bacteria, viruses, lipopolysaccharide (LPS) and cytokines including GM-CSF, IL-1, IL-2 and IFN-.gamma..
TNF-.alpha. interacts with two different receptors, TNF receptor I (TNFRI, p55) and TNFRII (p75), in order to transduce its effects, the net result of which is altered gene expression. Cellular factors induced by TNF-.alpha. include interleukin-1 (IL-1), interleukin-6 (IL-6), interleukin-8 (IL-8), interferon-.gamma. (IFN-.gamma.), platelet derived growth factor (PDGF) and epidermal growth factor (EGF), and endothelial cell adhesion molecules including endothelial leukocyte adhesion molecule 1 (ELAM-1), intercellular adhesion molecule-I (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) (Tracey, K. J., et al., Annu. Rev. Cell Biol., 1993, 9, 317-343; Arvin, B., et al., Ann. NY Acad. Sci., 1995, 765, 62-71).
Despite the protective effects of the cytokine, overexpression of TNF-.alpha. often results in disease states, particularly in infectious, inflammatory and autoimmune diseases. This process may involve the apoptotic pathways (Ksontini, R., et al., J. Immunol., 1998, 160, 4082-4089).
High levels of plasma TNF-.alpha. have been found in infectious diseases such as sepsis syndrome, bacterial meningitis, cerebral malaria, and AIDS; autoimmune diseases such as rheumatoid arthritis, inflammatory bowel disease (including Crohn's disease), sarcoidosis, multiple sclerosis, Kawasaki syndrome, graft-versus-host disease and transplant (allograft) rejection; and organ failure conditions such as adult respiratory distress syndrome, congestive heart failure, acute liver failure and myocardial infarction (Eigler, A., et al., Immunol. Today, 1997, 18, 487-492). Other diseases in which TNF-.alpha. is involved include asthma (Shah, A., et al., Clinical and Experimental Allergy, 1995, 25, 1038-1044), brain injury following ischemia (Arvin, B, et al., Ann. NY Acad. Sci., 1995, 765, 62-71), non-insulin-dependent diabetes mellitus (Hotamisligil, G. S., et al., Science, 1993, 259, 87-90), insulin-dependent diabetes mellitus (Yang, X.-D., et al., J. Exp. Med., 1994, 180, 995-1004), hepatitis (Ksontini, R, et al., J. Immunol., 1998, 160, 4082-4089), atopic dermatitis (Sumimoto, S., et al., Arch. Dis. Child., 1992, 67, 277-279), and pancreatitis (Norman, J. G., et al., Surgery, 1996, 120, 515-521). Further, inhibitors of TNF-.alpha. have been suggested to be useful for cancer prevention (Suganuma, M., et al. (Cancer Res., 1996, 56, 3711-3715). Elevated TNF-.alpha. expression may also play a role in obesity (Kern, P. A., J. Nutr., 1997, 127, 1917S-1922S). TNF-.alpha. was found to be expressed in human adipocytes and increased expression, in general, correlated with obesity.
There are currently several approaches to inhibiting TNF-.alpha. expression. Approaches used to treat rheumatoid arthritis include a chimeric anti-TNF-.alpha. antibody, a humanized monoclonal anti-TNF-.alpha. antibody, and recombinant human soluble TNF-.alpha. receptor (Camussi, G., Drugs, 1998, 55, 613-620). Other examples are indirect TNF-.alpha. inhibitors including phosphodiesterase inhibitors (e.g. pentoxifylline) and metalloprotease inhibitors (Eigler, A, et al., Immunol. Today, 1997, 18, 487-492). An additional class of direct TNF-.alpha. inhibitors is oligonucleotides, including triplex-forming oligonucleotides, ribozymes, and antisense oligonucleotides.
Several publications describe the use of oligonucleotides targeting TNF-.alpha. by non-antisense mechanisms. U.S. Pat. No. 5,650,316, WO 95/33493 and Aggarwal, B. B. et al. (Cancer Research, 1996, 56, 5156-5164) disclose triplex-forming oligonucleotides targeting TNF-.alpha.. WO 95/32628 discloses triplex-forming oligonucleotides especially those possessing one or more stretches of guanosine residues capable of forming secondary structure. WO 94/10301 discloses ribozyme compounds active against TNF-.alpha. mRNA. WO 95/23225 discloses enzymatic nucleic acid molecules active against TNF-.alpha. mRNA.
A number of publications have described the use of antisense oligonucleotides targeting nucleic acids encoding TNF-.alpha.. The TNF-.alpha. gene has four exons and three introns. WO 93/09813 discloses TNF-.alpha. antisense oligonucleotides conjugated to a radioactive moiety, including sequences targeted to the 5'-UTR, AUG start site, exon 1, and exon 4 including the stop codon of human TNF-.alpha.. EP 0 414 607 B1 discloses antisense oligonucleotides targeting the AUG start codon of human TNF-.alpha.. WO 95/00103 claims antisense oligonucleotides to human TNF-.alpha. including sequences targeted to exon 1 including the AUG start site. Hartmann, G. et al. (Mol. Med., 1996, 2, 429-438) disclose uniform phosphorothioates and mixed backbone phosphorothioate/phosphodiester oligonucleotides targeted to the AUG start site of human TNF-.alpha.. Hartmann, G et al. (Antisense Nucleic Acid Drug Devel., 1996, 6, 291-299) disclose antisense phosphorothioate oligonucleotides targeted to the AUG start site, the exon 1/intron 1 junction, and exon 4 of human TNF-.alpha.. d'Hellencourt, C. F. et al. (Biochim. Biophys. Acta, 1996, 1317, 168-174) designed and tested a series of unmodified oligonucleotides targeted to the 5'-UTR, and exon 1, including the AUG start site, of human TNF-.alpha.. Additionally, one oligonucleotide each was targeted to exon 4 and the 3'-UTR of human TNF-.alpha. and one oligonucleotide was targeted to the AUG start site of mouse TNF-.alpha.. Rojanasakul, Y. et al. (J. Biol. Chem., 1997, 272, 3910-3914) disclose an antisense phosphorothioate oligonucleotide targeted to the AUG start site of mouse TNF-.alpha.. Taylor, M. F. et al. (J. Biol. Chem., 1996, 271, 17445-17452 and Antisense Nucleic Acid Drug Devel., 1998, 8, 199-205) disclose morpholino, methyl-morpholino, phosphodiester and phosphorothioate oligonucleotides targeted to the 5'-UTR and AUG start codon of mouse TNF-.alpha.. Tu, G.-C. et al. (J. Biol. Chem., 1998, 273, 25125-25131) designed and tested 42 phosphorothioate oligonucleotides targeting sequences throughout the rat TNF-.alpha. gene.
Interestingly, some phosphorothioate oligodeoxynucleotides have been found to enhance lipopolysaccharide-stimulated TNF-.alpha. synthesis up to four fold due to nonspecific immunostimulatory effects (Hartmann et al. Mol. Med., 1996, 2, 429-438).
Accordingly, there remains an unmet need for therapeutic compositions and methods for inhibiting expression of TNF-.alpha., and disease processes associated therewith.